On a generalized Levinthal's paradox: The role of long- and short range interactions in complex bio-molecular reactions, including protein and DNA folding.

The current protein folding literature is reviewed. Two main approaches to the problem of folding were selected for this review: geometrical and biophysical. The geometrical approach allows the formulation of topological restrictions on folding, that are usually not taken into account in the construction of physical models. In particular, the topological constraints do not allow the known funnel-like energy landscape modeling, although most common methods of resolving the paradox are based on this method. The very paradox is based on the fact that complex molecules must reach their native conformations (complexes that result from reactions) in an exponentially long time, which clearly contradicts the observed experimental data. In this respect we considered the complexity of the reactions between ligands and proteins. On this general basis, the folding-reaction paradox was reformulated and generalized. We conclude that prospects for solving the paradox should be associated with incorporating a topology aspect in biophysical models of protein folding, through the construction of hybrid models. However, such models should explicitly include long-range force fields and local cell biological conditions, such as structured water complexes and photon/phonon/soliton waves, ordered in discrete frequency bands. In this framework, collective and coherent oscillations in, and between, macromolecules are instrumental in inducing intra- and intercellular resonance, serving as an integral guiding network of life communication: the electrome aspect of the cell. Yet, to identify the actual mechanisms underlying the bonds between molecules (atoms), it will be necessary to perform dedicated experiments to more definitely solve the particular time paradox.

Inhibition of renal rho kinase attenuates ischemia/reperfusion-induced injury.

The Rho kinase pathway plays an important role in dedifferentiation of epithelial cells and infiltration of inflammatory cells. For testing of the hypothesis that blockade of this cascade within the kidneys might be beneficial in the treatment of renal injury the Rho kinase inhibitor, Y27632 was coupled to lysozyme, a low molecular weight protein that is filtered through the glomerulus and is reabsorbed in proximal tubular cells. Pharmacokinetic studies with Y27632-lysozyme confirmed that the conjugate rapidly and extensively accumulated in the kidney. Treatment with Y27632-lysozyme substantially inhibited ischemia/reperfusion-induced tubular damage, indicated by reduced staining of the dedifferentiation markers kidney injury molecule 1 and vimentin, and increased E-cadherin relative to controls. Rho kinase activation was inhibited by Y27632-lysozyme within tubular cells and the interstitium. Y27632-lysozyme also inhibited inflammation and fibrogenesis, indicated by a reduction in gene expression of monocyte chemoattractant protein 1, procollagen Ialpha1, TGF-beta1, tissue inhibitor of metalloproteinase 1, and alpha-smooth muscle actin. Immunohistochemistry revealed reduced macrophage infiltration and decreased expression of alpha-smooth muscle actin, collagen I, collagen III, and fibronectin. In contrast, unconjugated Y27632 did not have these beneficial effects but instead caused systemic adverse effects, such as leukopenia. Neither treatment improved renal function in the bilateral ischemia/reperfusion model. In conclusion, the renally targeted Y27632-lysozyme conjugate strongly inhibits tubular damage, inflammation, and fibrogenesis induced by ischemia/reperfusion injury.

Cell-specific delivery of a transforming growth factor-beta type I receptor kinase inhibitor to proximal tubular cells for the treatment of renal fibrosis.

PURPOSE: Activation of tubular epithelial cells by transforming growth factor-beta (TGF-beta) plays an important role in the pathogenesis of renal tubulointerstitial fibrosis. We developed a renally accumulating conjugate of a TGF-beta type-I receptor kinase inhibitor (TKI) and evaluated its efficacy in vitro and in vivo. METHODS: TKI was conjugated to the protein Lysozyme (LZM) via a platinum-based linker. TKI-LZM was evaluated in human tubular cells (HK-2) for its anti-fibrotic activity. Plasma, kidney and urine drug levels after a single intravenous dose of TKI-LZM in rats were determined by HPLC or immunodetection. Anti-fibrotic effects of TKI-LZM were examined in the unilateral ureteral obstruction (UUO) model. RESULTS: TKI-LZM conjugate was successfully synthesized at an 1:1 drug/carrier ratio, and inhibited TGF-beta1-induced procollagen-1alpha1 gene expression in HK-2 cells. In vivo, TKI-LZM accumulated rapidly in tubular cells and provided a local depot for 3 days. Interestingly, a single dose of TKI-LZM inhibited the activation of tubular cells and fibroblasts in UUO rats and reduced renal inflammation. In contrast, free TKI at an equimolar (low) dosage exhibited little effects. CONCLUSIONS: Inhibition of TGF-beta signaling by local drug delivery is a promising antifibrotic strategy, and demonstrated the important role of tubular activation in renal fibrosis.

Targeting of stabilized plasmid lipid particles to hepatocytes in vivo by means of coupled lactoferrin.

For non-viral gene delivery we prepared stabilized plasmid lipid particles (SPLPs), to which lactoferrin (LF) was coupled as a hepatocyte specific targeting ligand. LF-SPLPs and untargeted SPLPs labeled with [3H]cholesteryloleyl-ether were injected into rats. About 87% of the LF-SPLPs were eliminated from the blood within 5 min, while 80% of untargeted SPLPs were still circulating after 2 h. Fifty-two percent of the LF-SPLPs were taken up by hepatocytes, while non-parenchymal liver cells accounted for 16% of the uptake. Despite the efficient targeting of LF-SPLPs to hepatocytes and their capacity to transfect HepG2 and COS-7 cells in vitro, expression of a reporter gene was not detected in vivo. Overall, covalent coupling of LF to SPLPs leads to massive delivery in hepatocytes after systemic administration. However, these LF-SPLPs are not able to transfect these cells in vivo.

A novel lipid-based drug carrier targeted to the non-parenchymal cells, including hepatic stellate cells, in the fibrotic livers of bile duct ligated rats.

In fibrotic livers, collagen producing hepatic stellate cells (HSC) represent a major target for antifibrotic therapies. We designed liposomes with surface-coupled mannose 6-phosphate (M6P) modified human serum albumin (HSA) to target HSC via the M6P receptor. In this study we determined the pharmacokinetics and target specificity of M6P-HSA-liposomes in a rat model of liver fibrosis. Ten minutes after injection of [(3)H]-M6P-HSA-liposomes 90% of the dose has cleared the circulation. The blood elimination of these liposomes was counteracted by free M6P-HSA and polyinosinic acid, a competitive inhibitor of scavenger receptors. The M6P-HSA-liposomes accumulated in HSC. However, also Kupffer cells and endothelial cells contributed to the uptake of M6P-HSA-liposomes in the fibrotic livers. Polyinosinic acid inhibited the accumulation of the liposomes in Kupffer cells and liver endothelial cells, but not in HSC. PCR analysis revealed that cultured HSC express scavenger receptors. This was confirmed by Western blotting, although activation of HSC diminishes scavenger receptor protein expression. In conclusion, in a rat model for liver fibrosis M6P-HSA-liposomes can be efficiently targeted to non-parenchymal cells, including HSC. M6P receptors and scavenger receptors are involved in the cellular recognition of these liposomes, allowing multiple pharmacological interference in different pathways involved in the fibrosis.

A phase I/IIa study with succinylated human serum albumin (Suc-HSA), a candidate HIV-1 fusion inhibitor.

BACKGROUND: Succinylated human serum albumin (Suc-HAS) is a negatively charged neo-glycoprotein that binds to the positively charged V3-loop of HIV-1 gp120, acting as HIV-1-fusion inhibitor in vitro (IC50: 0.5-5.0 microg/ml). Suc-HSA was safe in rats and monkeys, and showed antiretroviral effect in a human-to-mouse model. We evaluated safety and pharmacokinetics of single and multiple doses of Suc-HSA in HIV-1-infected individuals. METHODS: First, six untreated, chronically HIV-1-infected patients were randomized to a single dose of 1 or 10 mg/kg Suc-HSA intravenously. Second, five consecutive daily doses (10 mg/kg, based on the results of the single dose study) were given to four patients. Safety laboratory assessments, Suc-HSA plasma levels, plasma HIV-1 RNA (pVL), and CD4+ T-cell counts were determined. RESULTS: Increase of liver transaminases (grade 1/2) occurred in one of six patients in the single-dose phase and in three of four patients in the multiple-dosing phase. Suc-HSA plasma levels were undetectable 4 h after a single dose of 1 mg/kg. After a dose of 10 mg/kg, plasma levels were more sustained, but declined under the target plasma concentration (10 microg/ml) 12-24 h post-dosing. After multiple dosing, plasma levels reached peak values 2h post-dosing as predicted by our kinetic model. However, trough levels were below the target concentrations. There was no change in pVL or CD4+ T-cell count in either the single- or multiple-dosing phase. CONCLUSIONS: At the chosen dosing regimens, adequate antiviral plasma levels were not maintained, probably because the hepatic clearance was more rapid than expected. This may partially explain the lack of effect on pVL and CD4+ T-cell count. The observed liver transaminase increases prohibit further dose escalation.

Local inhibition of liver fibrosis by specific delivery of a platelet-derived growth factor kinase inhibitor to hepatic stellate cells.

Liver fibrosis is characterized by excessive proliferation and activation of hepatic stellate cells (HSC), a process in which platelet-derived growth factor (PDGF) plays an important role. Inhibition of liver fibrosis via specific delivery of a PDGF kinase inhibitor to HSC might therefore be an attractive strategy. The HSC-selective carrier mannose-6-phosphate modified human serum albumin (M6PHSA) was equipped with a tyrosine kinase inhibitor, 4-chloro-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide (PAP19) (an imatinib derivative), by means of the platinum-based universal linkage system (ULS). The antifibrotic activity of PAP19-M6PHSA was evaluated in culture-activated rat HSC and precision-cut liver slices from fibrotic rats. After 24-h incubation, both free inhibitor PAP19 and PAP19-M6PHSA showed potent activity, as determined by quantitative reverse transcription-polymerase chain reaction analysis of alpha-smooth muscle actin (alphaSMA) and procollagen 1a1. Next, we examined the organ distribution and antifibrotic activity of PAP19-M6PHSA in bile duct-ligated (BDL) rats. Male Wistar rats at day 10 after BDL were administered a single dose of PAP19-M6PHSA and sacrificed at 2 h, 1 day, or 2 days afterward. The accumulation of PAP19-M6PHSA in the liver was quantified by high-performance liquid chromatography analysis (30% of the injected dose at 2 h) and detected in the liver by staining of the carrier. Liver drug levels were sustained at 24 and 48 h after the single dose. Furthermore, PAP19-M6PHSA reduced collagen deposition (Sirius red staining) and alphaSMA staining of activated HSC at these time points in comparison with saline-treated rats. We therefore conclude that delivery of a PDGF-kinase inhibitor to HSC is a promising technology to attenuate liver fibrogenesis.

Delivery of viral vectors to hepatic stellate cells in fibrotic livers using HVJ envelopes fused with targeted liposomes.

Hepatic stellate cells (HSC) are a major target for antifibrotic therapies in the liver and in particular gene delivery to these cells would be relevant. Previously, we demonstrated that mannose 6-phosphate human serum albumin (M6P-HSA) coupled liposomes accumulate in HSC in fibrotic livers. Here we prepared a M6P-HSA modified viral vector that allows the targeted delivery of plasmid DNA to HSC. Therefore, UV inactivated hemagglutinating virus of Japan (HVJ) containing plasmid DNA was fused with M6P-HSA liposomes to yield HVJ liposomes targeted to HSC. These new particles had a diameter of approximately 200 nm, as determined by electron microscopy. In a carbon tetrachloride mouse model of liver fibrosis, M6P-HSA-HVJ-liposomes associated with HSC. In conclusion, our results demonstrate that fusion of M6P-HSA liposomes with HVJ envelopes results in HVJ particles that accumulate in HSC, allowing for new possibilities to interfere with fibrosis in the liver.

Effects of a new bioactive lipid-based drug carrier on cultured hepatic stellate cells and liver fibrosis in bile duct-ligated rats.

In the fibrotic liver, hepatic stellate cells (HSC) produce large amounts of collagen and secrete variety of mediators that promote development of fibrosis in this organ. Therefore, these cells are considered an attractive target for antifibrotic therapies. We incorporated the bioactive lipid dilinoleoylphosphatidylcholine (DLPC) into the membrane of liposomes, and then we evaluated its effect on hepatic stellate cell activation and liver fibrosis. To target DLPC-liposomes to HSC, human serum albumin modified with mannose 6-phosphate (M6P-HSA) was coupled to the surface of these liposomes. In vitro, the effects of the carrier were determined in primary cultures of HSC, Kupffer cells, and liver endothelial cells using real-time reverse transcription-polymerase chain reaction. In vivo DLPC-liposomes were tested in bile duct-ligated rats. Targeted M6P-HSA-DLPC-liposomes and DLPC-liposomes significantly reduced gene expression levels for collagen 1alpha1, alpha-smooth muscle actin (alpha-SMA), and transforming growth factor-beta (TGF-beta) in cultured HSC. In fibrotic livers, DLPC-liposomes decreased gene expression for TGF-beta and collagen 1alpha1 as well as alpha-SMA and collagen protein expression. In contrast, M6P-HSA-DLPC-liposomes enhanced expression of profibrotic and proinflammatory genes in vivo. In cultured Kupffer and endothelial cells M6P-HSA liposomes influenced the expression of proinflammatory genes. Both types of liposomes increased hepatocyte glycogen content in fibrotic livers, indicating improved functionality of the hepatocytes. We conclude that DLPC-containing liposomes attenuate activation of cultured HSC. In fibrotic livers, M6P-HSA-mediated activation of Kupffer and endothelial cells probably counteracts this beneficial effect of DLPC-liposomes. Therefore, these bioactive drug carriers modulate the activity of all liver cells during liver fibrosis.

Precision-cut fibrotic rat liver slices as a new model to test the effects of anti-fibrotic drugs in vitro.

BACKGROUND/AIMS: Current cell culture models contributed significantly to the study of liver fibrosis and the testing of anti-fibrotic drugs but mimic the complex in vivo milieu poorly. Therefore, we evaluated fibrotic rat liver slices as a new, more physiologic in vitro model to test anti-fibrotic compounds. METHODS: Precision-cut slices (8 mm diameter, 250 microm thickness) were prepared from fibrotic rat livers three weeks after bile-duct ligation and incubated for 0-48 h, during which viability and progression of the fibrotic process was evaluated. In addition, the effects of pentoxifylline, gleevec, and dexamethasone on mRNA expression of markers for fibrosis were determined. RESULTS: Fibrotic liver slices remained viable during 48 h of incubation, with increasing alphaSMA and pro-collagen 1a1 mRNA expression, and alphaSMA and collagen protein content after prolonged incubation. Addition of pentoxifylline, gleevec, or dexamethasone during incubation dose-dependently inhibited pro-collagen-1a1 and alphaSMA mRNA expression after 24h of incubation without influencing slice viability. CONCLUSIONS: Fibrotic liver slices are a promising tool to test anti-fibrotic drugs in vitro in a multicellular, fibrotic milieu, which cannot be achieved in vitro using other models. Importantly, this method may provide the opportunity to study anti-fibrotic compounds not only in animal but also in fibrotic human liver tissue.

Intracellular delivery of the p38 mitogen-activated protein kinase inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] in renal tubular cells: a novel strategy to treat renal fibrosis.

During renal injury, activation of p38 mitogen-activated protein kinase (MAPK) in proximal tubular cells plays an important role in the inflammatory events that eventually lead to renal fibrosis. We hypothesized that local inhibition of p38 within these cells may be an interesting approach for the treatment of renal fibrosis. To effectuate this, we developed a renal-specific conjugate of the p38 inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] and the carrier lysozyme. First, we demonstrated that SB202190 inhibited the expression of albumin-induced proinflammatory (monocyte chemoattractant protein-1) and transforming growth factor (TGF)-beta1-induced profibrotic (procollagen-Ialpha1) genes over 50% in renal tubular cells (normal rat kidney-52E). Next, we conjugated SB202190 via a carbamate linkage to lysozyme. However, this conjugate rapidly released the drug upon incubation in serum. Therefore, we applied a new platinum(II)-based linker approach, the so-called universal linkage system (ULS), which forms a coordinative bond with SB202190. The SB202190-ULS-lysozyme remained stable in serum but released the drug in kidney homogenates. SB202190-ULS-lysozyme accumulated efficiently in renal tubular cells and provided a local drug reservoir during a period of 3 days after a single intravenous injection. Treatment with SB202190-ULS-lysozyme inhibited TGF-beta1-induced gene expression for procollagen-Ialpha1 by 64% in HK-2 cells. Lastly, we evaluated the efficacy of a single dose of the conjugate in the unilateral renal ischemia-reperfusion rat model. A reduction of intrarenal p38 phosphorylation and alpha-smooth muscle actin protein expression was observed 4 days after the ischemia-reperfusion injury. In conclusion, we have developed a novel strategy for local delivery of the p38 MAPK inhibitor SB202190, which may be of use in the treatment of renal fibrosis.

Selective targeting of pentoxifylline to hepatic stellate cells using a novel platinum-based linker technology.

Targeting of antifibrotic drugs to hepatic stellate cells (HSC) is a promising strategy to block fibrotic processes leading to liver cirrhosis. For this purpose, we utilized the neo-glycoprotein mannose-6-phosphate-albumin (M6PHSA) that accumulates efficiently in HSC during liver fibrosis. Pentoxifylline (PTX), an antifibrotic compound that inhibits HSC proliferation and activation in vitro, was conjugated to M6PHSA. We employed a new type of platinum-based linker, which conjugates PTX via coordination chemistry rather than via covalent linkage. When incubated in plasma or in the presence of thiol compounds, free PTX was released from PTX-M6PHSA at a sustained slow rate. PTX-M6PHSA displayed pharmacological activity in cultured HSC as evidenced by changes in cell morphology and reduction of collagen I production. PTX-M6PHSA and platinum coupled PTX did not induce platinum-related toxicity (Alamar Blue viability assay) or apoptosis (caspase activation and TUNEL staining). In vivo distribution studies in fibrotic rats demonstrated specific accumulation of the conjugate in nonparenchymal cells in the fibrotic liver. In conclusion, we have developed PTX-M6PHSA employing a novel type of platinum linker, which allows sustained delivery of the drug to HSC in the fibrotic liver.

Gliotoxin non-selectively induces apoptosis in fibrotic and normal livers.

BACKGROUND: Liver fibrosis is the common response to chronic liver injury, ultimately leading to cirrhosis. Several lines of evidence indicate that inducing apoptosis of hepatic stellate cells (HSC) may lead to regression of liver fibrosis. Recently, it was shown that gliotoxin (GTX) induces apoptosis of HSC. However, the clinical use of GTX may be limited because of the lack of cell and tissue specificity, causing a high risk of potentially severe adverse effects. The aim of this study, therefore, was to study the effect of GTX on different cells of the liver. METHODS: We used normal and fibrotic precision-cut rat liver slices to study the effect of GTX on the various resident liver cell types. In these slices, the complex cell-cell interactions are preserved, which closely mimics the in vivo situation. RESULTS: GTX exhibited a potent apoptosis-inducing activity in these slices. Both immunohistochemical stainings and real-time mRNA techniques showed that this apoptosis-inducing effect was seen in HSC. However, Kupffer cells and liver endothelial cells were also affected by GTX, whereas hepatocytes were only mildly affected. CONCLUSIONS: This study indicates that the apoptosis-inducing strategy to treat liver fibrosis has high potential, but it will be necessary to develop an HSC-specific therapy to prevent adverse effects.

The antiproliferative drug doxorubicin inhibits liver fibrosis in bile duct-ligated rats and can be selectively delivered to hepatic stellate cells in vivo.

Hepatic stellate cell (HSC) proliferation is a key event in liver fibrosis; therefore, pharmacological intervention with antiproliferative drugs may result in antifibrotic effects. In this article, the antiproliferative effect of three cytostatic drugs was tested in cultured rat HSC. Subsequently, the antifibrotic potential of the most potent drug was evaluated in vivo. As a strategy to overcome drug-related toxicity, we additionally studied how to deliver this drug specifically to HSC by conjugating it to the HSC-selective drug carrier mannose-6-phosphate-modified human serum albumin (M6PHSA). We investigated the effect of cisplatin, chlorambucil, and doxorubicin (DOX) on 5-bromo-2'-deoxyuridine incorporation in cultured HSC and found DOX to be the most potent drug. Treatment of bile duct-ligated (BDL) rats with daily i.v. injections of 0.35 mg/kg DOX from day 3 to 10 after BDL reduced alpha-smooth muscle actin-stained area in liver sections from 8.5 +/- 0.8 to 5.1 +/- 0.9% (P < 0.01) and collagen-stained area from 13.1 +/- 1.3 to 8.9 +/- 1.5% (P < 0.05). DOX was coupled to M6PHSA, and the organ distribution of this construct (M6PHSA-DOX) was investigated. Twenty minutes after i.v. administration, 50 +/- 6% of the dose was present in the livers, and colocalization of M6PHSA-DOX with HSC markers was observed. In addition, in vitro studies showed selective binding of M6PHSA-DOX to activated HSC. Moreover, M6PHSA-DOX strongly attenuated HSC proliferation in vitro, indicating that active drug is released after uptake of the conjugate. DOX inhibits liver fibrosis in BDL rats, and HSC-selective targeting of this drug is possible. This may offer perspectives for the application of antiproliferative drugs for antifibrotic purposes.

On the role and fate of LPS-dephosphorylating activity in the rat liver.

Gut-derived lipopolysaccharide (LPS) plays a role in the pathogenesis of liver diseases like fibrosis. The enzyme alkaline phosphatase (AP) is present in, among others, the intestinal wall and liver and has been previously shown to dephosphorylate LPS. Therefore, we investigated the effect of LPS on hepatic AP expression and the effect of AP on LPS-induced hepatocyte responses. LPS-dephosphorylating activity was expressed at the hepatocyte canalicular membrane in normal and fibrotic animals. In addition to this, fibrotic animals also displayed high LPS-dephosphorylating activity around bile ducts. The enzyme was shown to dephosphorylate LPS from several bacterial species. LPS itself rapidly enhanced the intrahepatic mRNA levels for this enzyme within 2 h by a factor of seven. Furthermore, in vitro and in vivo studies showed that exogenous intestinal AP quickly bound to the asialoglycoprotein receptor on hepatocytes. This intestinal isoform significantly attenuated LPS-induced hepatic tumor necrosis factor-alpha and nitric oxide (nitrite and nitrate) responses in vitro. The enzyme also reduced LPS-induced hepatic glycogenolysis in vivo. This study shows that LPS enhances AP expression in hepatocytes and that intestinal AP is rapidly taken up by these same cells, leading to an attenuation of LPS-induced responses in vivo. Gut-derived LPS-dephosphorylating activity or enzyme upregulation within hepatocytes by LPS may therefore be a protective mechanism within the liver.

Prediction of the pharmacokinetics of succinylated human serum albumin in man from in vivo disposition data in animals and in vitro liver slice incubations.

Suc-HSA is a potent HIV-inhibitor with possible application in man. To facilitate the assessment of dosing regimens for future phase I clinical studies, we predicted the pharmacokinetic properties of Suc-HSA in man. Slices prepared from rat, monkey and human liver were incubated with succinylated albumin, and the maximum uptake rate V(m) and Michaelis-Menten constant K(m) were calculated. The pharmacokinetics after multiple doses of Suc-HSA were studied in rats. The pharmacokinetic parameters of Suc-HSA in man were predicted from the results and data from literature, using pharmacokinetic modeling and interspecies scaling techniques, and potential intravenous dose regimens for HIV treatment in man were calculated. On the basis of in vitro uptake studies in rat, monkey and human liver slices and in vivo disposition data in monkey (data from earlier study) and rat, we predicted the following parameters for liver uptake in humans: V(m) 82.5 microg h(-1) kg(-1) and K(m) 0.228 microg ml(-1). The predicted steady-state concentration after daily intravenous bolus doses of 1 mg kg(-1) is between 4 and 30 microg ml(-1), i.e. well above the IC50 of about 0.4 microg ml(-1). Additional loading doses of 8 mg kg(-1) in total are needed to reach steady-state within a few days.

Interaction of targeted liposomes with primary cultured hepatic stellate cells: Involvement of multiple receptor systems.

BACKGROUND/AIMS: In designing a versatile liposomal drug carrier to hepatic stellate cells (HSC), the interaction of mannose 6-phosphate human serum albumin (M6P-HSA) liposomes with cultured cells was studied. METHODS: M6P-HSA was covalently coupled to the liposomal surface and the uptake and binding of 3H-labelled M6P-HSA liposomes by primary rat HSC and liver endothelial cells was determined. The targeting ability of M6P-HSA liposomes to HSC was tested in bile duct ligated rats using immunohistochemical methods. RESULTS: The association of M6P-HSA liposomes with HSC was 4-fold higher than of control liposomes. An excess of M6P-HSA inhibited this association by 58%, indicating M6P receptor specificity. The scavenger receptor competitor polyinosinic acid abolished association of M6P-HSA liposomes with HSC. M6P-HSA liposomes also amply associated with endothelial cells, which abundantly express scavenger receptors. Endocytosis of M6P-HSA liposomes by HSC was temperature dependent and could be inhibited by monensin. In the fibrotic liver M6P-HSA liposomes co-localised with HSC. CONCLUSIONS: Coupling of M6P-HSA to liposomes strongly increases the in vitro uptake of these liposomes by HSC and endothelial cells. Both the mannose 6-phosphate receptor and the scavenger receptors are involved in the uptake process. M6P-HSA liposomes are potential drug carriers to HSC in the fibrotic liver.

Cell-specific targeting of lipid-based carriers for ODN and DNA.

It is well recognized that there is an urgent need for non-toxic systemically applicable vectors for biologically active nucleotides to fully exploit the current potential of molecular medicine in gene therapy. Cell-specific targeting of non-viral lipid-based carriers for ODN and DNA is a prerequisite to attain the concentration of nucleic acids required for therapeutic efficacy in the target tissue. In this review we will address the most promising approaches to selective targeting of liposomal nucleic acid carriers in vivo. In addition, the routes of entry and intracellular processing of these carrier systems are discussed as well as physiological factors potentially interfering with the biological and/or therapeutic activity of their nucleotide pay-load.

Bioanalysis and pharmacokinetics of the p38 MAPkinase inhibitor SB202190 in rats.

We have developed a sensitive and reproducible high performance liquid chromatography (HPLC)-UV method for the quantification of the p38 MAPkinase inhibitor SB202190 in serum, kidney homogenates and urine samples. Liquid-liquid extraction of SB202190 from the samples was performed using diethylether after adding a derivative of SB202190 as internal standard (I.S.). Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of acetonitrile-water-trifluoroacetic acid (30:70:0.1, v/v/v; pH 2.0). Both drug and I.S. were measured at 350 nm and eluted at 5.0 and 10.6 min, respectively. Peak-height ratios of the drug and the I.S. were used for the quantification of SB202190 from the different matrixes. The limit of quantitation of SB202190 in serum, kidney and urine were 0.25 microg/ml, 1 microg/g and 1 microg/ml, respectively. The average recoveries were 74, 75 and 92% in serum, kidney and urine, respectively. The intra- and inter-day precision (% CV) and accuracy (% bias) were below 15% for all concentrations. The method was successfully applied for a pharmacokinetic study of SB202190 in rats.